Alpha-adrenergic stimulation is known to lead to the formation of at least two second messangers, inositol trisphosphate (IP3) which is thought to release Ca2+ from the sarcoplasmic reticulum (SR) and 1,2 diacylglycerol (DAG) which activates a Ca2+-dependent protein kinase, i.e. protein kinase C (PKC). Previous work done in our laboratory showed that both DAG and the tumor promoting agent phorbol ester increase membrane activation of PKC and have a negative inotropic effect in adult cardiac myocytes. We hypothesized that because of its Ca2+-dependence that during high cell Ca2+ loading PKC activation would be more marked than under normal cell Ca2+ loading and possibly determine a negative inotropic effect of alpha-adrenergic stimulation rather than the better known positive response which could be related to the action of IP3 to release Ca2+ from the SR. Isolated rat ventricular myocytes, pretreated with propranolol were used to investigate the effect of alpha-adrenergic stimulation with phenylephrine on the frequency of spontaneous contractile waves (CW), which represent the mechanical expression of spontaneous Ca2+ release from the SR, and on twitch amplitude (TA). CW were measured in the unstimulated state and TA was determine during field stimulation. a. In the absence of stimulation, in 5 mM Cao treatment with phenylephrine led to a significant reduction in CW frequency and this was reversible upon removal of the drug. b. In cells stimulated to contract at a rate of 0.2 Hz, in d mM Cao CW appeared in some of the diastolic intervals. Treatment with phenylephrine caused abolition of diastolic CW and a significant diminution in TA. This negative effect of phenylephrine was in contrast to a diminution in TA. This negative effect of phenylephrine was in contrast to a positive inotropic action observed in parallel studies in 1 mM Ca. These findings are consistent with the view that the effect of alpha-adrenergic stimulation of cardiac cells can vary in relation to intracellular (Ca2+).